1. Field of the Invention (Technical Field)
The present invention relates to the detection and measurement of the ability of sperm to undergo the acrosome reaction, and thereby permits determination of the fertility of male mammals in general.
2. Description of the Related Art, Including Information Disclosed under 37 C.F.R. Sections 1.97-1.99 (Background Art)
With the development of new and effective techniques of birth control, attention has become more focused on the converse problem, infertility. One out of six couples of child bearing age is affected by infertility, and about 40% of these cases are related to problems in the male. Female infertility problems have been systematically addressed for a number of years, but there are relatively few clinical tests relating to male infertility and its causes, other than an examination of sperm count, sperm morphology, and presence of anti-sperm antibodies. S. A. Carson et al (1988) "Antibody binding patterns in infertile males and females as detected by immunobead test, gel-agglutination test, and sperm immobilization test" Fertility and Sterility 49:487-492 and T. F. Kruger et al (1988) "Predictive value of abnormal sperm morphology in in vitro fertilization" Fertility and Sterility 49:112-117. In clinical assessment of male infertility, most of the standard tests currently performed in semen analysis detect only the most obvious defects. More recent developments include computer assisted semen analysis (CASA), the hamster ova penetration assay (SPA), and the hypo-osmotic swelling tests (HOS). The value of these kinds of tests for predicting fertility of patients is still being analyzed. The ultimate test of human sperm fertility is its ability to penetrate the human oocyte. The functional defects underlying individual infertility problems in many cases remain undiscovered.
In attempting to determine the causes of male infertility, physicians examine spermatozoa for their number, morphology, and forward motility characteristics. They also look for the presence of anti-sperm antibodies which may interfere with fertilization. Although these tests may be marginally useful, they detect only the most obvious defects of sperm, and only a small percentage of infertility cases are attributable to gross defects or to antibodies. Thus, the value of these kinds of tests in predicting potential fertility of patients is questionable. Many cases of unexplained infertility exist in which the above parameters are all within normal limits, yet sperm are incapable of fertilizing an ovum. The standard tests currently performed in semen analysis do not address sperm function in a direct manner. The functional defects underlying individual infertility problems in many cases remain undiscovered. In addition, the tests are time consuming and somewhat expensive to perform, requiring highly trained technologists and expensive equipment.
An exception to this generalization is the sperm-cervical mucus interaction test (SCMI), which examines the ability of a husband's sperm to penetrate his partner's cervical mucus, a critical step in the transport of sperm to the site of fertilization. G. N. Clarke et al (1988) "Sperm antibodies and human in vitro fertilization" Fertility and Sterility 49:1018-1025. This test of sperm function has only recently been performed on a routine basis and is useful in assessing certain types of infertility. Another functional test developed recently is the sperm penetration assay (SPA), in which sperm are examined for their ability to penetrate hamster ova which have been denuded of their zona pellucida. S. L. Corson et al (1988) "The human sperm-hamster egg penetration assay: prognostic value" Fertility and Sterility 49:328-334. The test has limited predictive value, since sperm are left in contact with the eggs for several hours, and zona-free ova are not physiologically relevant to actual fertilization; in fact, penetration of the zona pellucida surrounding the ovum is one of the critical steps in the fertilization process. D. P. Wolf et al (1985) "Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies" Biology of Reproduction 32:1157-1162. Both tests, the SCMI and SPA, require highly trained technologists, well-equipped laboratories, and several hours to perform.
Human male infertility can be attributed to two basic types of dysfunctions; those which are intrinsic to the sperm and result in defective sperm or sperm production, and those which are extrinsic to sperm, such as antibodies, which affect sperm function as a result of their interaction with sperm. Both types of defects may inhibit the acrosome reaction. The acrosome reaction is a critical process by which sperm release hydrolytic enzymes that degrade the zona pellucida, a thick protein and polysaccharide protective coating that surrounds the ovum. Penetration of the zona enables the sperm and ovum to come into contact, fuse, and complete the fertilization process. W. Byrd and D. P. Wolf (1988) "Acrosomal status in fresh and capacitated human ejaculated sperm" Biology of Reproduction 34:859-869; A. H. Sathananthan and C. Chen (1986) "Sperm-oocyte membrane fusion in the human during monospermic fertilization" Gamete Research 15:177-186.
The acrosome reaction is thought to be stimulated by contact with, or binding to, the zona pellucida, which induces a rise in the intracellular calcium level of the sperm. The rise in intracellular calcium in turn triggers the release of the hydrolytic enzymes, which leads to degradation of zona proteins. The ability to undergo the acrosome reaction and penetrate the zona pellucida is an absolute requirement for fertilization. Sperm may be defective in this ability as the result of a genetic defect, such as in round-headed sperm syndrome in which the acrosome is absent, or as the result of extrinsic factors such as sperm-coating antibodies which bind the sperm and prevent release of acrosomal contents. Currently, these defects can only be detected by electron microscopy or immunofluorescence and radioimmunoassay.
Spermatozoa undergo two steps in the acrosome reaction process: a reversible capacitation step followed by the irreversible acrosome reaction. The acrosome reaction enables sperm to penetrate the acellular glycoprotein layer, the zona pellucida, of the ovum. J. M. Bedford (1970) "Sperm Capacitation and Fertilization in Mammals" Biol Reprod (Suppl) 2:128-158. Capacitation in vivo is a little understood process that occurs to the sperm over a several hour period in the female reproductive tract after entry into the uterus. It is thought to involve, among other things, the removal of protein factors present in the seminal plasma which adhere to the sperm surface and prevent premature release of the acrosome. In research procedures, sperm can be capitated by incubation in physiologic saline for at least four hours. After this time, 30% to 50% of sperm undergo the acrosome reaction upon addition of calcium and its appropriate ionophore.
The ability of human spermatozoa to undergo the acrosome reaction is critical to the fertilization process. A variety of assays have been developed; however, each presents significant limitations. M. A. Lee, et al (1987) "Capacitation and acrosome reactions in human spermatozoa monitored by a chlortetracycline fluorescence assay" Fertility and Sterility 48:649-658; S. S. Suarez et al (1986) "Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid" Gamete Research 14:107-121; F. A. Feuchter et al (1987), "The human sperm acrosome reaction: involvement of a vimentin-associated surface glycoprotein of the equatorial segment" Anatomical Record 218:44A; N. L. Cross et al (1986) "Two simple methods for detecting acrosome-reacted human sperm" Gamete Research 15:213-226; and, M. Kallajoki et al (1986) "The fate of acrosomal staining during the acrosome reaction of human spermatozoa as revealed by a monoclonal antibody and PNA-lectin" Int J Andrology 9:181-194. Each assay requires a significant period of time to perform, particularly induction of capacitation, and no assay has been reduced to a simple dot-blot format.
Other methods of in vitro capacitation of sperm have been studied. Glycosaminoglycans have been shown to accelerate conversion of proacrosin to acrosin, thought to be a step in the induction of the acrosome reaction. R. F. Parrish et al (1980) "Glycosaminoglycan stimulation of the in vitro conversion of boar proacrosin into acrosin" J Androl 1:89-95.
A mixture of proteolytic enzymes, containing trypsin, chymotrypsin and .beta.-amylase, has been used to strip the surface coat of rabbit sperm and induce capacitation in vitro. P. V. Dandekar and M. Gordon (1974) "Electron microscope evaluation of rabbit eggs exposed to spermatozoa treated with capacitating agents." J Reprod Fert 44:143-146. Unlike human sperm, rabbit sperm do not capacitate well in physiologic saline and must be enzyme-treated before a significant number will acrosome react when mixed with rabbit ova.
It is generally assumed that elevation of calcium ions in the cytoplasm between the plasma and outer acrosomal membranes is the key event in initiation of the acrosomal reaction. Capacitation thus involves the preparation of the sperm for the elevation in calcium ions. One method used for several years in mammalian sperm research employs a calcium ionophore to induce the acrosome reaction. This method mimics the natural process in which contact with the zona pellucida induces a rise in the intracellular calcium level of sperm, which in turn triggers the acrosome reaction. The ionophore is effective at inducing the acrosome reaction in properly capacitated and fertile sperm, but has no effect on oligozoospermic and otherwise infertile samples. R. J. Aitken (1984) Analysis of Human Sperm Function Following Exposure to the Ionophore A23187: Comparison of Normospermic and Oligozoospermic Men.
Once the acrosome reaction has been induced, there are a number of markers which can be used to detect and measure the percentage of spermatozoa undergoing the acrosome reaction. Lectin molecules can be used as markers of the human acrosome. Peanut agglutinin (PNA) and Pisum sativum agglutinin (PSA) are now recognized as markers of the acrosomal region of human sperm. Cross, supra, and Kallajoki, supra. PSA binds specifically to acrosomal contents, and PNA binds either to acrosomal contents or acrosomal membranes. Only acrosome intact sperm bind to the lectins, and sperm that have undergone the acrosome reaction do not bind the lectins. On sperm which are capable of undergoing the acrosome reaction, the acrosomal markers are shed and can be identified in the supernatant solution; on those which are incapable of the reaction, the markers are retained (or if genetically absent, no markers are shed) and are not identifiable in the supernatant solution. PNA appears to be pan-specific in its reactivity with the acrosome, that is, it can be used in species other than human.
Acrosomal staining can also be accomplished by the methods of J. H. D. Bryan and S. R. Akruk (1977) "A Naphthol Yellow S and Erythrosin B Staining Procedure for use in Studies of the Acrosome Reaction of Rabbit Spermatozoa" Stain Technol 52:47-50. In addition, methods have been developed for staining using monoclonal antibodies specific for acrosomal markers. Kallajoki, supra. Monoclonal antibodies are highly specific markers, and are useful for some techniques. However, they are not without limitations when used for probes of the acrosome reaction. Monoclonal antibodies are species specific, and thus lack pan-species applicability. In addition, monoclonal antibodies often exhibit low avidity, and not infrequently have a short shelf-life. Monoclonal antibodies are expensive to produce, and must be obtained from specialized laboratories. Other methods are also known to those skilled in the art.
R. L. Ax and R. W. Lenz, U.S. Pat. No. 4,767,703, Method for Assessing the Fertility of Male Mammals used a glycosaminoglycan to induce an acrosome reaction in sperm. A representative sample of incubated sperm from both the individual to be tested and a control are then stained and counted by means of observation by light microscopy to measure the increase in acrosome reaction in the test portion as compared to the control portion. This method requires a lengthy incubation of sperm in the glycosaminoglycan to induce the acrosome reaction; nine to twenty-two hours incubation are reported in the method.
J. A. Houghton, Patent Cooperation Treaty International Patent Application No. WO89/02743 (filed Sep. 23, 1988), In Vitro Method for the Induction of the Spermatozoal Acrosome Reaction and Application of Said Method to the Assessment of Spermatozoa and the Treatment of Male-Related Infertility subjects spermatozoa to electropermeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of calcium ions. This method thus requires specialized equipment to induce the reaction. The method also induces a reaction even in sperm incapable of naturally undergoing the acrosome reaction, and thus is of limited utility in any diagnostic assay.
There is no known method of conducting both a microscopic and dot blot assay to examine the ability of human spermatozoa to undergo the acrosome reaction. These assays address a function of sperm that is critical to the fertilization process and which is not currently tested in clinical laboratories.